The present invention relates to improved methods for producing a compound of interest. Numerous approaches have been applied to date in generating strains for protein over-expression and/or production. This includes, but is not limited to, making strains with multi-copies of the gene encoding the compound of interest and applying strong promoter sequences.
Each specific amino acid is encoded by a minimum of one codon and a maximum of six codons. Prior research has shown that codon usage in genes encoding the cell's polypeptides is biased among species (Kanaya, S, Y. Yamada, Y. Kudo and T. Ikemura (1999) Studies of codon usage and tRNA genes at 18 unicellular organisms and quantification of Bacillus subtilis tRNAs: gene expression level and species-specific diversity of codon usage based on multivariate analysis. Gene 238:143-155). Prior publications disclose optimization of codon use in a given host cell to improve polypeptide production (as example see WO 97/11086). More specifically, WO 03/70957 describes optimized codon use in filamentous fungi for producing plant polypeptides. In all these cases of ‘classic’ codon optimization, a native codon has been substituted by the most frequent codon from a reference set of genes, whereas the rate of codon translation for each amino acid is designed to be high (optimized). However, this ‘classic’ codon optimization neglects the other codons for which tRNAs are still available.
Recently, in WO 03/85114 a harmonization of codon use was described which takes into effect the distribution of optimal and non-optimal codons in genes, assuming that these effect protein folding. Using this method of harmonization of codon use for a gene results in the substitution of good (bad) codons in the donor organism by good (bad) ones in the host organism. However, this method of codon harmonization (WO 03/085114) neglects non-optimal codons since they are not replaced by more optimal ones. Additionally, the method cannot be applied to homologous genes. Another publication describes an additional way to improve polypeptide production in a host cell by using an improved consensus translational initiator sequence (U.S. Pat. No. 6,461,837 B1); the consensus sequence 5′-nyCnnhCACC(ATG)-3′ (SEQ ID NO: 36) is claimed.
There is still a need for improved methods for producing a polypeptide in a filamentous fungal cell.